The advent of recombinant DNA techniques has made it possible to select single protein components with interesting properties and produce them on a large scale. This represents an improvement over the previously employed production process using microorganisms isolated from nature and producing a mixture of proteins which would either be used as such or separated after the production step.
Since the traditional methods were rather time-consuming, more rapid and less cumbersome methods were developed.
A such technique is described in WO 93/11249 (Novo Nordisk A/S).
The method described in WO 93/11249 comprises the steps of:
a) cloning, in suitable vectors, a DNA library from an organism suspected of producing one or more proteins of interest; PA1 b) transforming suitable yeast host cells with said vectors; PA1 c) culturing the host cells under suitable conditions to express any protein of interest encoding by a clone in the DNA library; and PA1 d) screening for positive clones by determining any activity of a protein expressed in step c). PA1 i) PCR amplification of said DNA with PCR primers with homology to (a) known gene(s) encoding a polypeptide with an activity of interest, PA1 ii) linking the obtained PCR product to a 5' structural gene sequence and a 3' structural gene sequence, PA1 iii) expressing said resulting hybrid DNA sequence, PA1 iv) screening for hybrid DNA sequences encoding a polypeptide with said activity of interest or related activity, PA1 v) isolating the hybrid DNA sequence identified in step iv)
According to this method it is necessary to prepare a DNA library, comprising complete genes encoding polypeptides with activities of interest. Such a library has traditionally been made on mRNA isolated from micro-organisms which has been cultivated and isolated.
As it is only possible with known methods to cultivate about 2% of the microorganisms known today (i.e. cultivable microorganisms), genes encoding polypeptides from a huge number of microorganisms (i.e. un-cultivable microorganisms) are generally difficult to identify and clone on the basis of screening technologies used today, such as the above mentioned.